臺灣博碩士論文加值系統

幾丁聚醣是幾丁質去乙醯化而成的帶正電多醣。醣胺多醣是帶負電的多醣體包括硫酸軟骨素、肝素以及玻尿酸可以調控骨生成的活性。本篇研究利用幾丁聚醣和戊二醛交聯成薄膜後，並在薄膜上覆蓋三種不同種類的醣胺多醣。本篇研究將7F2骨母細胞及RAW264.7巨噬細胞培養在幾丁聚醣與醣胺多醣交聯的薄膜上
，探討此薄膜對骨細胞的影響。雖然幾丁聚醣的薄膜具高度疏水性，但幾丁聚醣與糖胺多醣交聯的薄膜卻有60-80%的細胞貼附率。在本篇研究中，幾丁聚醣薄膜經肝素修飾後，可以回復因為幾丁聚醣薄膜而下降的鹼性磷酸酶活性及提升OPG/RANKL的比值。而在抗發炎的實驗中，幾丁聚醣與糖胺多醣交聯的薄膜均可抑制一氧化氮的產生以及COX-2與MMP-3發炎因子的表現。幾丁聚醣的薄膜或是經肝素、玻尿酸覆蓋後皆可抑制抗酒石酸酸性磷酸酶的活性，而經玻尿酸修飾後的抑制效果又比幾丁聚醣薄膜更佳。經由這些實驗結果，我們知道幾丁聚醣的薄膜會降低骨生成的活性但可以抑制骨吸收，但在糖胺多醣的修飾後可以部份地回復骨生成的活性。在未來的實驗中，幾丁聚醣及糖胺多醣不只可應用在生物材料上，或許也可以用來治療骨質疏鬆症。

Chitosan is a cationic polysaccharide derived from the partial deacetylation of chitin. Hyaluronic acid (HA), chondroitin sulfate (CS) and Heparin (HP) are anionic glycosaminoglycan (GAGs) which can regulate osteogenic activity. In this study, chitosan films were prepared by glutaraldehyde crosslinking reaction and then composited with three different types of GAGs. 7F2 osteoblast-like cells and macrophages Raw264.7 were studied in vitro for the effect of chitosan films and complexes with GAGs on osteometabolism. Although chitosan films are highly hydrophobic, the films associated with GAG-chitosan complexes have showed about 60-80% cell attachment. Furthermore, chitosan complexes with HP could increase ALP activity, up-regulate OPG/RANKL mRNA ratio and inhibit COX-2 mRNA expression in comparison with chitosan films only. All chitosan films and complexes with GAGs could inhibit MMP-3 mRNA expression of 7F2 osteoblasts under the IL-1βstimulation. On the other side, three types of the GAG-chitosan complexes could significantly inhibit LPS induced-Nitric Oxide expression. In addition, chitosan complexes with HP and HA can down-regulate Tartrate-resistant acid phosphatase (TRAP) activity but not CS-chitosan complexes. Based on these results, we conclude that chitosan complexes with HP can increase ALP activity and up-regulate OPG/RANKL ratio in osteoblasts and chitosan complexes with HP and HA could reduce TRAP activity in osteoclasts.

Abstract……………………………………………………………………………….2
中文摘要………………………………………………………………………………3
致謝……………………………………………………………………………………4
Index…………………………………………………………………………………..5
Chapter 1 Introduction
1.1 Bone remodeling…………………………………………………………….7
1.2 Osteoarthritis………………………………………………………………..8
1.3 Arthroplasty…………………………………………………………………9
1.4 Chitosan…………………………………………………………………...10
1.5Glycosaminoglycans………………………………………………………..10
1.6 Chondroitin sulfate……………………………………………………….11
1.7 Heparin……………………………………………………………………..12
1.8 Hyaluronic acid…………………………………………………………….13
1.9 Aim of the Thesis…………………………………………………………..13
Chapter 2 Methods
2.1 Materials and cell culture…………………………………………………15
2.2 The preparation of chitosan films………………………………………...15
2.3 Cell viability and Proliferation assay………………………………………….16
2.4 Cell attachment……………………………………………………………16
2.5 Contact angle………………………………………………………………16
2.6 Nitrite assay………………………………………………………………..17
2.7 Alkaline phosphate (ALP) activity………………………………………..18
2.8 Tartate-resistant acid phosphate (TRAP) activity……………………....18
2.9 Quantitative real-time PCR………………………………………………19
2.10 Statistical analysis……………………………………………………….20
Chapter 3 Results
3.1 The effect of glycosaminoglycan on the cell viability and proliferation..21
3.2 The effect of glycosaminoglycans and chitosan on ALP activity and NO production in LPS-Induced Macrophages…………………………….…....24
3.3 The effect of glycosaminglycan and protein coatings on contact angles of chitosan films……………………………………………………………..….26
3.4 Cell attachment on chitosan films and complexes with different glycosaminoglycan coatings………………………………………..….…….27
3.5 The effect of chitosan films and complexes with different glycosaminoglycans on ALP assay…………………………………….…....29
3.6 The effect of chitosan films and complexes with different glycosaminoglycans on NO production in LPS-Induced Macrophages…..29
3.7 The chitosan films and the complexes could inhibit osteoclast differentiation in RAW264.7 macrophages…………………………………30
3.8 The effect of chitosan films and complexes with different glycosaminoglycans on 7F2 osteoblasts gene expression…………………..32
Chapter 4 Discussion…………………………………………………………...…35
Chapter 5 Conclusion………………………………………………….………….39
Reference……………………………………………………………………………41

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